Hydroxyurea with AKT2 inhibition decreases vaso-occlusive events in sickle cell disease mice

Abstract

Key Points• Coadministration of HU and an AKT2 inhibitor has beneficial effects on acute vaso-occlusive events and survival in SCD mice.Heterotypic cell-cell adhesion and aggregation mediate vaso-occlusive events in patients with sickle cell disease (SCD). Although hydroxyurea (HU), an inducer of fetal hemoglobin, is the main therapy for treatment of SCD, it is unclear whether it has immediate benefits in acute vaso-occlusive events in SCD patients. Using real-time fluorescence intravital microscopy, we demonstrated that short-term coadministration of HU and Akti XII, an AKT2 inhibitor, efficiently reduced neutrophil adhesion and platelet-neutrophil aggregation in venules of Berkeley (SCD) mice challenged with tumor necrosis factor a (TNF-a) or hypoxia/reoxygenation. Importantly, compared with HU or Akti XII treatment alone, short-term treatment with both agents significantly improved survival in those mice. We found that the level of plasma nitric oxide species was elevated by HU but not Akti XII, AKT2 phosphorylation levels in activated neutrophils and platelets were reduced by Akti XII but not HU, and the expression of endothelial E-selectin and intercellular adhesion molecule 1 was decreased by either agent. Our results suggest that short-term coadministration of HU and Akti XII has immediate benefits for acute vaso-occlusive events and survival in SCD mice exceeding those seen for single therapy. (Blood. 2015;126(22):2511-2517 IntroductionSickle cell disease (SCD), an inherited blood disorder, results from a homozygous mutation at the 6th position (Glu6Val) of the b-globin chain (hemoglobin S [HbS]) or from compound heterozygous forms such as HbSC and HbS-b-thalassemia.1 HbS tends to polymerize in the deoxygenated state, and this leads to the sickling and hemolysis of red blood cells.2,3 Importantly, decreased nitric oxide (NO) bioavailability and increased oxidative stress contribute to the pathophysiology of SCD, including activation of endothelial cells (ECs), inflammation, and organ damage. 4 Recurrent vaso-occlusive events, the hallmark of SCD, are mediated by adhesion and aggregation of red blood cells, leukocytes, and platelets on activated ECs 5,6 and cause pain crises in SCD patients. 7 Hydroxyurea (HU), the only drug approved by the US Food and Drug Administration for treatment of SCD, stimulates HbF production,8 serves as an NO donor, 9,10 and inhibits tissue factor expression in leukocytes.11 Although the mechanism by which HU acts is still not fully understood, previous studies showed that treatment of SCD patients with HU is associated with the production of NO and increases HbF levels in an NO-dependent manner. 9,10 Studies that use a humanized SCD (Berkeley) mouse model have consistently shown that short-term treatment with HU can have immediate benefits in vaso-occlusive events, independently of HbF production. 12The authors further demonstrated that combination therapy of HU and a phosphodiesterase 9 inhibitor efficiently inhibits acute vasoocclusion in SCD mice. 12Intravital microscopic ...

ARQ 092, an orally-available, selective AKT inhibitor, attenuates neutrophil-platelet interactions in sickle cell disease

“…13 We have shown that AKT2 positively regulates the function of αMb2 integrin and P-selectin during vascular inflammation 12 and that combining hydroxyurea with AKT2 inhibition has immediate benefits in acute vaso-occlusive events and improves survival in SCD mice. 9 Although these results suggest that AKT2 inhibition may be a supplemental therapy for SCD patients with vaso-occlusive crises, no AKT2-specific inhibitor is currently available in the clinic.…”

“…SCD mice (20-24 weeks old) were generated by transplantation of bone marrow cells isolated from Berkeley mice into lethally irradiated wildtype mice as described previously. 9,29 Three to 4 months after transplantation, polymerase chain reaction and electrophoresis analyses showed that all chimeric mice expressed the transgene (human HbS) (Online Supplementary Figure S1). In contrast, mouse hemoglobin was not detected.…”

“…[5][6][7] Consistently, in vivo studies showed that intravenous infusion of hydroxyurea increases the level of plasma NO metabolites (NOx) and has beneficial effects on vaso-occlusive events in Berkeley mice, a model of SCD. 8,9 However, SCD patients on hydroxyurea therapy often suffer from vaso-occlusive crises, suggesting that a novel or supplemental therapy is required.…”

“…[9][10][11][12] Among several receptors and counter-receptors, the neutrophil-platelet association is primarily mediated by the interaction of neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) and αMb2 integrin with platelet Pselectin and glycoprotein Ibα (GPIbα), respectively. 13 We have shown that AKT2 positively regulates the function of αMb2 integrin and P-selectin during vascular inflammation 12 and that combining hydroxyurea with AKT2 inhibition has immediate benefits in acute vaso-occlusive events and improves survival in SCD mice.…”

“…8,9,29 Compared to the vehicle controls (saline, S; 0.01 M phosphoric acid, PA), treatment with hydroxyurea or both hydroxyurea and ARQ 092 significantly prolonged survival times in TNF-α-challenged SCD mice, whereas treatment with ARQ 092 alone did not improve survival (S versus hydroxyurea, P=0.0028; PA versus hydroxyurea + ARQ 092, P=0.0017; ARQ 092 versus hydroxyurea + ARQ 092, P=0.0087; and hydroxyurea versus hydroxyurea + ARQ 092, P=0.31) ( Figure 4F). Fifty percent of SCD mice treated with vehicle (S), hydroxyurea, vehicle (PA), ARQ 092, and both hydroxy urea and ARQ 092 died at 4.0, 5.5, 4.0, 4.1, and >6 hours, respectively, after TNF-α injection.…”